The invention relates to flow cytometers which are valuable tools for mechanistic studies of molecular interactions, such as cell function.
Flow cytometry allows particle associated fluorescence to be measured in comparison to fluorescence from the surrounding media which contributes very little to the measured signal. Cell associated fluorescence can be measured in a background of free fluorescent indicator. This is a valuable feature when determining ligand or dissociation kinetics where the cells must exist in a fluorescent milieu during analysis. In addition, flow cytometry allows for the examination of individual cells for kinetic analysis of selected populations in an heterogeneous mixture of cells. Bulk technologies such as fluorometry measure population average fluorescence and do not always distinguish free from bound fluorescence.
Early time measurements in conventional flow cytometers are limited by the time required to remove the sample tube from the instrument, inject reagent, manually mix the components return the sample tube and re-establish flow. This sequence usually requires a minimum of 7-10 seconds. Because of this limitation, the use of flow cytometry in the acquisition of kinetic data has been limited to processes with relatively slow kinetics. Many of the biological and biophysical processes of interest occur in less than five seconds. For example, in the study of receptor-ligand interactions, ligand binding affinity is calculated in part by determining the disassociation kinetics of the ligand. When the Kd of binding is in the microM range, off rates are sufficiently fast that they often cannot be measured by traditional flow cytometry.
Previously, sample handling systems designed to shorten this delay, have been explored (see FIGS. 1 and 2). These systems can be broadly classified into two groups: "time-window" and "time-zero" devices. Time-window systems continually analyze cells at a single time point after reagent mixing. To achieve a kinetic sequence, many individual measurements must be made, adjusting the sample delivery apparatus between measurements. The first such system was built around a simple mixing "T". The sample was injected from one side of the "T" and reagent from the other. Both sample and reagent were delivered at a constant slow rate, which together provided sample to the flow cell at a rate slow enough to allow stable laminar flow. The time point being measured was a function of the distance between the "T" and the laser interrogation point. Another approach using coaxial mixing has recently been reported. Coaxial mixing is a system in which sample is injected into the middle of a flowing stream of reagent. Once again, the time point measured is a function of the distance of the sample insertion tube from the laser interrogation point. These systems are useful when collecting large amounts of data at a single time point. However, they suffer when attempting to analyze a dynamic sequence of events.
Time-zero devices, on the other hand, mix sample and reagent at a single time point and follow the sequence of events over time. A stir-bar based sample mixing and injection system has been used by several groups. Stir-bar based systems employ a sample tube containing the cell suspension. Reagent is injected into the tube, mixed with a magnetic stir-bar and pushed to the flow cytometer laser interrogation point. Although one-second delivery times have been reported, it remains to be demonstrated that cells and reagent can be thoroughly mixed before arrival at the laser interrogation point. Mixing is more difficult in these systems because of the relatively large volumes used.
More recently, a syringe driven system has been described, in a published article, that has been shown to thoroughly mix small volumes in a reaction "T" and deliver the mixed sample to the laser in 300 mSec ("Rapid Mix Flow Cytometry with Subsecond Kinetic Resolution", Nolan et al., Cytometry 21, pp. 223-229, 1995.) Two features of this system significantly enhanced its ability to acquire data at very early time points. First, a valve is positioned proximal to the flow-cell sample input port. This allowed large volumes of mixed sample to be pushed quickly to that valve while shunting the excess volume to waste. The valve port is then changed directing the mixed sample to the flow cell at a much slower rate, easily accommodated by the flow cell orifice. Second, stable fluorescence measurements were improved by eliminating events that did not flow through the laser focal point by gating only on those events with an optimal forward light scatter signal. As seen in FIG. 2, cells not flowing through the center of the laser will receive less illumination, because of the gaussian intensity profile of a focused laser and because of the shorter distance across the beam when off-center. Therefore, cells forced outside the center of the laser beam by turbulent flow will have both a reduced light scatter signal as well as reduced fluorescence.
It is an object of the present invention to produce a syringe driven system without the interference of turbulent flow.
It is a further object of the present invention to provide an automated sample delivery system yielding maximum flexibility to accommodate a variety of applications.
It is a further object of the present invention to obtain particles in focus at the earliest time after the sample has been injected.
It is a further object of the present invention to control sheath flow in a flow cytometer to enhance and clarify particle analysis.